DNA Technology and Genomics

DNA Technology and Genomics
Wilson Muse Schoolcraft College

12.1 Genes can be cloned in recombinant plasmids
12.1 Genes can be cloned in recombinant plasmids Genetic engineering involves manipulating genes for practical purposes Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA Recombinant DNA is formed by joining DNA sequences from two different sources One source contains the gene that will be cloned Another source is a gene carrier, called a vector Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Figure 12.1 is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Referring to this figure in class helps students relate the text to your lecture. 2. The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to cut and remove the minute of film and a way to cut the new film apart and insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic relationship.

Steps in cloning a gene Plasmid DNA is isolated DNA containing the gene of interest is isolated Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle DNA with the target gene is treated with the same enzyme and many fragments are produced Plasmid and target DNA are mixed and associate with each other At step 6, there are actually three types of products that include plasmid DNA: (1) The ends of the plasmid can rejoin so that its original sequence is restored. (2) A recombinant DNA molecule can be formed containing part or all of the gene of interest. (3) Recombinant DNA molecules form that contain sequences unrelated to the gene of interest, representing the largest percentage of recombinant molecules. This mixture of products is typically used to transform bacteria under conditions where each cell is likely to take up only one plasmid. The cells are grown to form colonies, and properties of the plasmid and target DNA are used to detect the colony containing the recombinant plasmid carrying the gene of interest. Plasmids usually contain marker genes whose products indicate the presence of the plasmid within a bacterial host. A common approach is to use a plasmid with two markers, genes whose products indicate the presence of the plasmid within a bacterial cell. The site at which the plasmid is cut to add the target DNA is within one of the marker genes. Bacterial cells that show the action of both marker genes are not carrying target DNA and can be eliminated from the population. Bacterial cells expressing only the intact marker gene carry a recombinant plasmid. If the whole genome from the target organism is represented, this collection of clones is called a gene library (see Module 12.3). DNA from cells in this library can be tested for hybridization to a probe (see Module 12.5), to identify the cell carrying the gene of interest. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Figure 12.1 is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Referring to this figure in class helps students relate the text to your lecture. 2. The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to cut and remove the minute of film and a way to cut the new film apart and insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic relationship.

12.1 Genes can be cloned in recombinant plasmids Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together The recombinant DNA is taken up by a bacterial cell The bacterial cell reproduces to form a clone of cells At step 6, there are actually three types of products that include plasmid DNA: (1) The ends of the plasmid can rejoin so that its original sequence is restored. (2) A recombinant DNA molecule can be formed containing part or all of the gene of interest. (3) Recombinant DNA molecules form that contain sequences unrelated to the gene of interest, representing the largest percentage of recombinant molecules. This mixture of products is typically used to transform bacteria under conditions where each cell is likely to take up only one plasmid. The cells are grown to form colonies, and properties of the plasmid and target DNA are used to detect the colony containing the recombinant plasmid carrying the gene of interest. Plasmids usually contain marker genes whose products indicate the presence of the plasmid within a bacterial host. A common approach is to use a plasmid with two markers, genes whose products indicate the presence of the plasmid within a bacterial cell. The site at which the plasmid is cut to add the target DNA is within one of the marker genes. Bacterial cells that show the action of both marker genes are not carrying target DNA and can be eliminated from the population. Bacterial cells expressing only the intact marker gene carry a recombinant plasmid. If the whole genome from the target organism is represented, this collection of clones is called a gene library (see Module 12.3). DNA from cells in this library can be tested for hybridization to a probe (see Module 12.5), to identify the cell carrying the gene of interest. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Figure 12.1 is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Referring to this figure in class helps students relate the text to your lecture. 2. The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to cut and remove the minute of film and a way to cut the new film apart and insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic relationship. Animation: Cloning a Gene

Figure 12.1 An overview of gene cloning.
E. coli bacterium Plasmid Cell with DNA containing gene of interest Bacterial chromosome 1 Isolate plasmid 2 Isolate DNA 3 Cut plasmid with enzyme DNA Gene of interest 4 Cut cell’s DNA with same enzyme Gene of interest 5 Combine targeted fragment and plasmid DNA Examples of gene use 6 Add DNA ligase, which closes the circle with covalent bonds Genes may be inserted into other organisms Recombinant DNA plasmid Gene of interest Figure 12.1 An overview of gene cloning. This figure shows steps 1–8 as detailed on the previous two slides. 9 Genes or proteins are isolated from the cloned bacterium 7 Put plasmid into bacterium by transformation Recombinant bacterium Harvested proteins may be used directly Examples of protein use 8 Allow bacterium to reproduce Clone of cells

E. coli bacterium Plasmid Cell with DNA containing gene of interest Isolate plasmid Bacterial chromosome 1 2 Isolate DNA DNA Gene of interest Figure 12.1 An overview of gene cloning.

E. coli bacterium Plasmid Cell with DNA containing gene of interest 1 Isolate plasmid Bacterial chromosome 2 Isolate DNA 3 Cut plasmid with enzyme DNA Gene of interest 4 Cut cell’s DNA with same enzyme Gene of interest Figure 12.1 An overview of gene cloning.

Combine targeted fragment and plasmid DNA
E. coli bacterium Plasmid Cell with DNA containing gene of interest Isolate plasmid Bacterial chromosome 1 2 Isolate DNA 3 Cut plasmid with enzyme DNA Gene of interest 4 Cut cell’s DNA with same enzyme Gene of interest 5 Combine targeted fragment and plasmid DNA Figure 12.1 An overview of gene cloning.

Combine targeted fragment and plasmid DNA
E. coli bacterium Plasmid Cell with DNA containing gene of interest 1 Isolate plasmid Bacterial chromosome 2 Isolate DNA 3 Cut plasmid with enzyme DNA Gene of interest 4 Cut cell’s DNA with same enzyme Gene of interest 5 Combine targeted fragment and plasmid DNA Figure 12.1 An overview of gene cloning. 6 Add DNA ligase, which closes the circle with covalent bonds Recombinant DNA plasmid Gene of interest

Recombinant DNA plasmid Gene of interest Put plasmid into bacterium
Recombinant DNA plasmid Gene of interest 7 Put plasmid into bacterium by transformation Recombinant bacterium Figure 12.1 An overview of gene cloning.

Recombinant DNA plasmid Gene of interest Put plasmid into bacterium
Recombinant DNA plasmid Gene of interest 7 Put plasmid into bacterium by transformation Recombinant bacterium Figure 12.1 An overview of gene cloning. 8 Allow bacterium to reproduce Clone of cells

Examples of gene use Genes may be inserted into other organisms
Examples of gene use Genes may be inserted into other organisms Recombinant DNA plasmid Gene of interest 9 Genes or proteins are isolated from the cloned bacterium 7 Put plasmid into bacterium by transformation Recombinant bacterium Harvested proteins may be used directly Figure 12.1 An overview of gene cloning. 8 Allow bacterium to reproduce Clone of cells Examples of protein use

12.2 Enzymes are used to “cut and paste” DNA
12.2 Enzymes are used to “cut and paste” DNA Restriction enzymes cut DNA at specific sequences Each enzyme binds to DNA at a different restriction site Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends” Fragments with complementary sticky ends can associate with each other, forming recombinant DNA DNA ligase joins DNA fragments together Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. The authors note the origin of the name restriction enzymes. In nature, these enzymes protect bacterial cells against foreign DNA. Thus, these enzymes “restrict” the invasion of foreign genetic material. 2. A genomic library of the sentence you are now reading would be all of the sentence fragments that made up the sentence. One could string together all of the words of this first sentence, without spaces between letters, and then conduct a word-processing edit, placing a space between any place where the letter e is followed by the letter n. The resulting fragments of this original sentence would look like this and would be similar to a genomic library. Age nomiclibraryofthese nte nceyouare nowreadingwouldbeallofthese nte ncefragme ntsthatmadeupthese nte nce. Animation: Restriction Enzymes

Restriction enzyme recognition sequence
DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other.

DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other.

DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Two (or more) fragments stick together by base-pairing Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other. 4

DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Two (or more) fragments stick together by base-pairing Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other. 4 DNA ligase pastes the strands Recombinant DNA molecule 5

12.3 Cloned genes can be stored in genomic libraries
A genomic library is a collection of all of the cloned DNA fragments from a target genome Genomic libraries can be constructed with different types of vectors Plasmid library: genomic DNA is carried by plasmids Phage library: genomic DNA is incorporated into bacteriophage DNA Bacterial artificial chromosome (BAC) library: specialized plasmids can carry large DNA sequences BAC libraries have been instrumental in decoding the human genome. Early BAC constructs were based on the F plasmid (see Module 10.23), but more recent versions have been customized for transfer and replication in specific applications. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. A genomic library of the sentence you are now reading would be all of the sentence fragments that made up the sentence. One could string together all of the words of this first sentence, without spaces between letters, and then conduct a word-processing edit, placing a space between any place where the letter e is followed by the letter n. The resulting fragments of this original sentence would look like this and would be similar to a genomic library. Age nomiclibraryofthese nte nceyouare nowreadingwouldbeallofthese nte ncefragme ntsthatmadeupthese nte nce.

Genome cut up with restriction enzyme Recombinant plasmid Recombinant
Recombinant plasmid Recombinant phage DNA or Figure 12.3 Genomic libraries. Bacterial clone Phage clone Plasmid library Phage library

12.4 Reverse transcriptase can help make genes for cloning
Complementary DNA (cDNA) is used to clone eukaryotic genes mRNA from a specific cell type is the template Reverse transcriptase produces a DNA strand from mRNA DNA polymerase produces the second DNA strand Advantages of cloning with cDNA Study genes responsible for specialized characteristics of a particular cell type Obtain gene sequences without introns Smaller size is easier to handle Allows expression in bacterial hosts Prokaryotic hosts are not capable of removing introns from transcripts of cloned eukaryotic genes. Therefore, cDNA cloning is used to isolate the exon-only portion of a gene so that a prokaryotic cell can accurately produce the gene product. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. A cDNA library is a way to learn what portion of the genome is active at any given time in a cell’s life. In a very general way, it is like looking at the list of books checked out at a school library (assuming that the checked-out books are being used). 2. Reverse transcriptase is introduced in Module 10.20, where HIV is discussed. Even if students were not assigned this chapter, Module provides a meaningful background for the natural and significant roles of this enzyme.

and addition of reverse transcriptase; synthesis of DNA strand
Cell nucleus Exon Intron Exon Intron Exon DNA of eukaryotic gene 1 Transcription RNA transcript 2 RNA splicing mRNA 3 Isolation of mRNA and addition of reverse transcriptase; synthesis of DNA strand Test tube Reverse transcriptase Figure 12.4 Making an intron-lacking gene from eukaryotic mRNA. Cells transcribe specific genes. Cells process transcripts, removing introns and splicing exons together, to produce mRNA. mRNA is isolated and used as a template to produce a single strand of complementary DNA. Reverse transcriptase catalyzes the reaction. mRNA is enzymatically removed. DNA polymerase synthesizes the second DNA strand using the first strand as a template. cDNA strand being synthesized 4 Breakdown of RNA 5 Synthesis of second DNA strand cDNA of gene (no introns)

12.5 Nucleic acid probes identify clones carrying specific genes
12.5 Nucleic acid probes identify clones carrying specific genes Nucleic acid probes bind to cloned DNA Probes can be DNA or RNA sequences complementary to a portion of the gene of interest A probe binds to a gene of interest by base pairing Probes are labeled with a radioactive isotope or fluorescent tag for detection Using a probe to locate a gene sequence has sometimes been compared to “finding a needle in a haystack.” If the gene of interest is the “needle,” the “haystack” represents the remaining gene and nongenic regions of DNA. A probe can be considered to be a “magnet” that will make it easier to find the needle. As with all analogies, this one has its limitations. In reality, the gene of interest and the remaining DNA sequences do not have significant chemical differences like metal and straw. But the crucial difference lies in the order of nucleotides, so that a probe complementary to the gene of interest will not bind to other DNA sequences. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Some Internet search programs rely upon a methodology similar in one way to the use of a nucleic acid probe. For example, if you want to find the lyrics to a particular song, but do not know the song title or artist, you might search the Internet using a unique phrase from the song. The search engine will scan millions of web pages to identify those sites containing that particular phrase. However, unlike a nucleic acid probe, you would search for the song by using a few of the lyrics. A nucleic acid probe search uses a sequence complementary to the desired sequence.

12.5 Nucleic acid probes identify clones carrying specific genes
Screening a gene library Bacterial clones are transferred to filter paper Cells are lysed and DNA is separated into single strands A solution containing the probe is added, and binding to the DNA of interest is detected The clone carrying the gene of interest is grown for further study Because cells are destroyed by the processes for probe binding and detection, screening is carried out on cells transferred to a filter. The pattern of cell growth on an agar plate is retained during the transfer so that the investigator can return to the plate if one of the clones is shown to have the DNA of interest. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Some Internet search programs rely upon a methodology similar in one way to the use of a nucleic acid probe. For example, if you want to find the lyrics to a particular song, but do not know the song title or artist, you might search the Internet using a unique phrase from the song. The search engine will scan millions of web pages to identify those sites containing that particular phrase. However, unlike a nucleic acid probe, you would search for the song by using a few of the lyrics. A nucleic acid probe search uses a sequence complementary to the desired sequence.

Radioactive DNA probe Mix with single- stranded DNA from
Radioactive DNA probe Mix with single- stranded DNA from genomic library Single-stranded DNA Figure 12.5 How a DNA probe tags a gene by base pairing. This figure shows the specific binding of the probe to the gene of interest due to complementary base pairing. Base pairing indicates the gene of interest

12.6 Recombinant cells and organisms can mass-produce gene products
Cells and organisms containing cloned genes are used to manufacture large quantities of gene products Capabilities of the host cell are matched to the characteristics of the desired product Prokaryotic host: E. coli Can produce eukaryotic proteins that do not require post-translational modification Has many advantages in gene transfer, cell growth, and quantity of protein production Can be engineered to secrete proteins Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. As noted in Module 12.6, DNA technology is primarily used to produce proteins. Challenge your students to explain why lipids and carbohydrates are not typically produced by these processes.

12.6 Recombinant cells and organisms can mass-produce gene products
Capabilities of the host cell are matched to the characteristics of the desired product Eukaryotic hosts Yeast: S. cerevisiae Can produce and secrete complex eukaryotic proteins Mammalian cells in culture Can attach sugars to form glycoproteins “Pharm” animals Will secrete gene product in milk Blood clotting Factor VIII is used to treat patients with hemophilia. This protein has 25 sites for carbohydrate attachment. Since glycosylation does not occur in prokaryotic or yeast cells, recombinant Factor VIII is produced in cultured mammalian (hamster) cells. For BLAST Animation Genetic Recombination in Bacteria, go to Animation and Video Files. Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. As noted in Module 12.6, DNA technology is primarily used to produce proteins. Challenge your students to explain why lipids and carbohydrates are not typically produced by these processes.

GM sheep Figure 12.6 “Pharm” animals that produce a human protein.
GM sheep Figure 12.6 “Pharm” animals that produce a human protein. Examples of products from “pharm” animals include: goats producing tissue plasminogen activator (TPA) for heart attack victims, sheep producing alpha-1-antitrypsin to treat emphysema, and pigs producing hemoglobin as a blood substitute.

Table 12.6 Some Protein Products of Recombinant DNA Technology.

DNA technology has changed the pharmaceutical industry and medicine
DNA technology has changed the pharmaceutical industry and medicine Products of DNA technology Therapeutic hormones Insulin to treat diabetes Human growth hormone to treat dwarfism Diagnosis and treatment of disease Testing for inherited diseases Detecting infectious agents such as HIV Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. Annual flu vaccinations are a common way to prevent diseases that cannot be easily treated. However, students might not understand why many people receive the vaccine every year. A new annual vaccine is necessary because the flu viruses keep evolving, another lesson in evolution that may be missed by your students.

Products of DNA technology
Products of DNA technology Vaccines Stimulate an immune response by injecting Protein from the surface of an infectious agent A harmless version of the infectious agent A harmless version of the smallpox virus containing genes from other infectious agents Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. Annual flu vaccinations are a common way to prevent diseases that cannot be easily treated. However, students might not understand why many people receive the vaccine every year. A new annual vaccine is necessary because the flu viruses keep evolving, another lesson in evolution that may be missed by your students.

Advantages of recombinant DNA products Identity to human protein
Purity Quantity Figure 12.7A Human insulin produced by bacteria.

Genetically modified organisms are transforming agriculture
Genetically modified (GM) organisms contain one or more genes introduced by artificial means Transgenic organisms contain at least one gene from another species GM plants Resistance to herbicides Resistance to pests Improved nutritional profile GM animals Improved qualities Production of proteins or therapeutics GM plants: Resistance to herbicides: Roundup Ready Soybeans contain a bacterial version of an amino acid synthesis enzyme that is less sensitive to glyphosate (Roundup). Resistance to pests: Bt corn produces an insect toxin, derived from the bacterium Bacillus thuringiensis. Improved nutritional profile: “Golden rice” has increased beta-carotene due to the presence of daffodil genes. GM animals: Improved qualities: Sheep with an extra copy of a growth hormone gene grow larger and faster and produce more milk and wool. Production of proteins or therapeutics: “Pharm” animals (see Module 12.6). The first GM organism approved for sale as food was the Flavr-Savr tomato. The modification was intended to prolong the shelf life of the tomato by keeping it from softening when ripe. Softening is caused by an enzyme called polygalacturonase that breaks down pectins in fruit. In the Flavr-Savr tomato, production of polygalacturonase was blocked by an antisense RNA molecule complementary to the enzyme’s mRNA. The shelf life of the tomato was indeed prolonged, but ultimately there was little flavor to savor as consumers found the fruit to have a bland taste! For Discovery Video Transgenics, go to Animation and Video Files. Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. Roundup Ready Corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMO), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms.

Agrobacterium tumefaciens
Agrobacterium tumefaciens DNA containing gene for desired trait 1 Ti plasmid Recombinant Ti plasmid Insertion of gene into plasmid Figure 12.8A Using the Ti plasmid as a vector for genetically engineering plants. A modified form of the Ti (tumor inducing) plasmid is used in plant genetic engineering. The tumor inducing genes have been removed from the plasmid but genes required for insertion into plant chromosomes have been retained. The gene of interest has been inserted into the plasmid under the control of a bacterial promoter. Restriction site

DNA containing gene for desired trait Plant cell 1 2 Ti plasmid Recombinant Ti plasmid Insertion of gene into plasmid Introduction into plant cells Figure 12.8A Using the Ti plasmid as a vector for genetically engineering plants. A modified form of the Ti (tumor inducing) plasmid is used in plant genetic engineering. The tumor inducing genes have been removed from the plasmid but genes required for insertion into plant chromosomes have been retained. The gene of interest has been inserted into the plasmid under the control of a bacterial promoter. DNA carrying new gene Restriction site

DNA containing gene for desired trait Plant cell 1 2 3 Ti plasmid Recombinant Ti plasmid Insertion of gene into plasmid Introduction into plant cells Regeneration of plant Figure 12.8A Using the Ti plasmid as a vector for genetically engineering plants. A modified form of the Ti (tumor inducing) plasmid is used in plant genetic engineering. The tumor inducing genes have been removed from the plasmid but genes required for insertion into plant chromosomes have been retained. The gene of interest has been inserted into the plasmid under the control of a bacterial promoter. DNA carrying new gene Plant with new trait Restriction site

Figure 12.8B A mix of “golden rice” and standard rice.

12.9 Genetically modified organisms raise concerns about human and environmental health
Scientists use safety measures to guard against production and release of new pathogens Concerns related to GM organisms Can introduce allergens into the food supply FDA requires evidence of safety before approval Exporters must identify GM organisms in food shipments May spread genes to closely related organisms Hybrids with native plants may be prevented by modifying GM plants Regulatory agencies address the safe use of biotechnology Bt corn (see notes for Module 12.8) has been the focus of two ecological concerns. The first is related to the development of resistance to Bt toxin by the European corn borer. Those insects that can survive the levels of Bt toxin produced by the corn will reproduce, and the level of resistance to the toxin will increase among their offspring. Bt corn has been formulated to provide a high dose of the toxin to eliminate the majority of corn borers that come into contact with it. In addition, farmers are required to provide a “refuge,” a field planted with non-Bt corn where susceptible corn borers can survive. The rationale is that these susceptible corn borers will interbreed with resistant ones that survive the Bt toxin, and the resulting offspring with lowered resistance will be subject to the high toxin levels from Bt corn. Farmers are concerned that the cost of planting a field where corn borers damage the crop will not be offset by the gains of planting Bt corn on the remaining fields. A second possible cause for concern is related to the spread of Bt corn pollen beyond the edges of a cornfield. A laboratory study showed 50% mortality for larvae of the monarch butterfly feeding on leaves of milkweed plants dusted with Bt corn pollen. A field study using potted milkweed plants at various distances from a Bt corn field showed 19% mortality for monarch larvae feeding on plants closest to the field. The extent to which monarch larvae encounter Bt pollen under natural conditions is not known, and studies of this phenomenon are continuing. Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. Roundup Ready Corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMO), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms.

Gene therapy may someday help treat a variety of diseases
Gene therapy aims to treat a disease by supplying a functional allele One possible procedure Clone the functional allele and insert it in a retroviral vector Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell Viral DNA and the functional allele will insert into the patient’s chromosome Return the cells to the patient for growth and division At the present time, gene therapy methods supply a functional allele but do not replace the defective one. Most currently used vectors promote random integration of a therapeutic allele into a chromosome, so the location may be far from the defective allele. The production of the functional gene product alleviates the disease symptoms. Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. As gene therapy technology expands, our ability to modify the genome in human embryos through in vitro fertilization permits genetic modification at the earliest stages of life. Future generations of humans, like our crops today, may include those with and without a genetically modified ancestry. The benefits and challenges of these technologies raise issues many students have never considered. Our students, and the generations soon to follow, will face the potential of directed human evolution.

Gene therapy may someday help treat a variety of diseases
SCID (severe combined immune deficiency) was the first disease treated by gene therapy First trial in 1990 was inconclusive Second trial in 2000 led to the development of leukemia in some patients due to the site of gene insertion Challenges Safe delivery to the area of the body affected by the disease Achieving a long-lasting therapeutic effect Addressing ethical questions In 1993, a gene therapy trial in the United States on three infants with SCID showed that the functional allele could be delivered to blood-forming umbilical cord stem cells and that those cells would repopulate the bone marrow when returned to the patient. Unfortunately, the stem cells did not generate a large enough population to completely alleviate the disease. In 2002, a group of Italian physicians achieved success with two SCID patients, treated at 7 and 30 months of age. Their approach was to reduce the population of bone marrow stem cells through chemical treatment prior to providing stem cells with the functional allele. The genetically corrected cells were able to substantially repopulate the bone marrow as a result. Both patients are healthy and growing and developing normally. Student Misconceptions and Concerns 1. The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. 2. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent provides proof of evolution that may be missed by your students. Teaching Tips 1. As gene therapy technology expands, our ability to modify the genome in human embryos through in vitro fertilization permits genetic modification at the earliest stages of life. Future generations of humans, like our crops today, may include those with and without a genetically modified ancestry. The benefits and challenges of these technologies raise issues many students have never considered. Our students, and the generations soon to follow, will face the potential of directed human evolution.

Cloned gene (normal allele) Insert normal gene into virus
1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Figure One type of gene therapy procedure. Bone marrow cell from patient Bone marrow 4 Inject cells into patient

DNA PROFILING similar to DNA Fingerprinting
Can be used to assign paternity or link DNA evidence to a crime scene

12.11 The analysis of genetic markers can produce a DNA profile
12.11 The analysis of genetic markers can produce a DNA profile DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual Compares genetic markers from noncoding regions that show variation between individuals Involves amplification (copying) of markers for analysis Sizes of amplified fragments are compared For BLAST Animation DNA Fingerprinting, go to Animation and Video Files. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. Teaching Tips 1. Figure describes the general steps of DNA profiling. This overview is a useful reference to employ while the details of each step are discussed.

Crime scene Suspect 1 Suspect 2 DNA isolated DNA of selected
Crime scene Suspect 1 Suspect 2 1 DNA isolated 2 DNA of selected markers amplified Figure An overview of DNA fingerprinting. This figure shows the process of DNA profiling (also called DNA fingerprinting). DNA is isolated. DNA is amplified by polymerase chain reaction (see Module 12.12). The amplified fragments are compared by agarose gel electrophoresis (see Module 12.13). 3 Amplified DNA compared

12.12 The PCR method is used to amplify DNA sequences
Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule Relies upon a pair of primers Short DNA molecules that bind to sequences at each end of the sequence to be copied Used as a starting point for DNA replication Repeated cycle of steps for PCR Sample is heated to separate DNA strands Sample is cooled and primer binds to specific target sequence Target sequence is copied with heat-stable DNA polymerase From one target DNA sequence, 30 cycles of PCR will produce over 1 million copies. The use of primers is related to the native activity of DNA polymerase. To synthesize a DNA strand, DNA polymerase adds nucleotides to the 3′ end of a short nucleotide strand bound to the template. In the cell, primers are composed of RNA, synthesized by an enzyme called primase. These RNA segments are later removed from the DNA product. In PCR, synthetically produced DNA primers serve as the starting point for the polymerase. The heat-stable Taq polymerase was isolated from Thermus aquaticus, a bacterium found in hot springs. The enzyme can withstand heating to 94oC and synthesize DNA at 72oC during PCR. This is a helpful illustration of the effect of natural selection in favoring a form of the enzyme that would not denature with the high temperatures of the bacterium’s environment. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive!

12.12 The PCR method is used to amplify DNA sequences
Advantages of PCR Can amplify DNA from a small sample Results are obtained rapidly Reaction is highly sensitive, copying only the target sequence Since PCR will amplify any DNA sequence with ends matching the primers, contamination by even a small amount of DNA that may contain primer-matching sites can be a concern. Stringent conditions are used for collecting and handling samples to guard against contamination. Critics of PCR point to possible contamination when questioning the accuracy of the method. The developer of PCR, Kary Mullis, was retained as an expert witness for the defense in the O. J. Simpson murder trial. While he was never called to testify, it was reported that he would cite contamination as one reason to discount the DNA evidence used in the trial. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive! see animation:

Figure 12.12 DNA amplification by PCR.
Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Genomic DNA 3 5 3 5 3 5 5 1 Heat to separate DNA strands 2 Cool to allow primers to form hydrogen bonds with ends of target sequences 3 DNA polymerase adds nucleotides to the 3 end of each primer 3 5 5 3 Target sequence 5 5 3 5 3 5 3 Figure DNA amplification by PCR. This figure shows several cycles of the PCR process, emphasizing that the number of templates doubles with each cycle. Primer New DNA

Cycle 1 yields 2 molecules Genomic DNA 3 5 3 5 3 5 5 DNA
Cycle 1 yields 2 molecules Genomic DNA 3 5 3 5 3 5 5 DNA polymerase adds nucleotides to the 3 end of each primer 1 Heat to separate DNA strands 2 Cool to allow primers to form hydrogen bonds with ends of target sequences 3 3 5 5 3 Target sequence 5 Figure DNA amplification by PCR. This figure shows several cycles of the PCR process, emphasizing that the number of templates doubles with each cycle. 5 3 5 3 5 3 Primer New DNA

Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules
Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Figure DNA amplification by PCR. This figure shows several cycles of the PCR process, emphasizing that the number of templates doubles with each cycle.

12.13 Gel electrophoresis sorts DNA molecules by size
Gel electrophoresis separates DNA molecules based on size DNA sample is placed at one end of a porous gel Current is applied and DNA molecules move from the negative electrode toward the positive electrode Shorter DNA fragments move through the gel pores more quickly and travel farther through the gel DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence Each band is a collection of DNA molecules of the same length For BLAST Animation Gel Electrophoresis, go to Animation and Video Files. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. Separating ink using paper chromatography is a simple experiment that approximates some of what occurs in gel electrophoresis. Consider doing this as a class demonstration while addressing electrophoresis. Cut a large piece of filter paper into a rectangle or square. Use markers to color large dots about 2 cm away from one the edge of the paper. Separate the dots from each other by 3–4 cm. Place the paper on edge, dots down, into a beaker containing about 1 cm of ethanol or isopropyl alcohol (50% or higher will do). The dots should not be in contact with the pool of alcohol in the bottom of the beaker. As the alcohol is drawn up the filter paper by capillary action, the alcohol will dissolve the ink dots. As the alcohol continues up the paper, the ink follows. Not all of the ink components move at the same speed, based upon their size and chemical properties. If you begin the process at the start of class, you should have some degree of separation by the end of a 50-minute period. Experiment with the technique a day or two before class to fine tune the demonstration. (Save and air-dry these samples for your class.) Consider using brown, green, and black markers, since these colors are often made by color combinations. Video: Biotechnology Lab

Mixture of DNA fragments of different sizes Longer (slower) molecules
Mixture of DNA fragments of different sizes Longer (slower) molecules Power source Gel Shorter (faster) molecules Figure Gel electrophoresis of DNA. Completed gel

12.14 STR analysis is commonly used for DNA profiling
Short tandem repeats (STRs) are genetic markers used in DNA profiling STRs are short DNA sequences that are repeated many times in a row at the same location The number of repeating units can differ between individuals STR analysis compares the lengths of STR sequences at specific regions of the genome Current standard for DNA profiling is to analyze 13 different STR sites D7S820 represents a region on chromosome 7 that is one of the 13 standard loci used for DNA profiling. Its sequence, GATA, can be repeated from 5 to 16 times. This results in 78 possible genotypes in the population. Sites with large amounts of variation are chosen for DNA profiling to have the greatest chance of distinguishing between individuals. Using more than one location allows a multiplication of the odds of finding two people with the same genotype. As described in Module 12.15, there should be a less than 1 in 10 billion chance that two unrelated individuals share the same profile for the 13 standard STR loci. This depends on the frequencies of the alleles in a specific population. For example, one DNA profiler analyzed his own DNA at the 13 STR sites and calculated that his profile would only be found among 1 in 7.7 quadrillion Caucasian individuals (1 in 7.7 x 1015). Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. In most legal cases, the probability of two people having identical DNA profiles can be one in 10 billion or more. However, eyewitness testimony has been a standard part of the justice system. If you want to make the point about the unreliability of eyewitnesses in a trial, compared to techniques such as genetic profiling, consider this exercise. Arrange for a person who is not well known to the class to run into your classroom, take something you have placed near you (perhaps a bag, stack of papers, or box), and leave quickly. You need to take care that no one in the class is so alarmed as to do something dangerous. Once the “thief” is gone, tell the class that this was planned and do not speak. Have them each write a description of the person, including height, hair color, clothing, facial hair, behavior, etc. Many students will be accurate, but some will likely get details wrong. This is also an effective exercise to demonstrate the need for large sample sizes and accurate recording devices for good scientific technique.

STR site 1 STR site 2 Crime scene DNA Number of short tandem
STR site 1 STR site 2 Crime scene DNA Number of short tandem repeats match Number of short tandem repeats do not match Suspect’s DNA Figure 12.14A Two representative STR sites from crime scene DNA samples. This figure shows differences in the numbers of short tandem repeats that can be used in DNA profiling.

Crime scene DNA Suspect’s DNA
Crime scene DNA Suspect’s DNA Figure 12.14B DNA profiles generated from the STRs in Figure 12.14A. This figure shows a comparison of fragments with different numbers of repeats by gel electrophoresis.

DNA profiling has provided evidence in many forensic investigations
Forensics Evidence to show guilt or innocence Establishing family relationships Paternity analysis Identification of human remains After tragedies such as the September 11, 2001, attack on the World Trade Center Species identification Evidence for sale of products from endangered species The stability of DNA provides substantial advantages in DNA profiling, as the following example shows. In San Diego, California, there was a case of an 8-year old girl who was abducted from her home and sexually abused. Police accused the girl’s father even though she claimed that a stranger had assaulted her. The father was arrested when the child changed her story, two years after the incident. As evidence was gathered to prepare for the father’s defense, a reexamination of the child’s nightgown revealed semen stains. The DNA pattern excluded the father and included a suspect who had been jailed on child molestation charges six weeks after the girl’s assault. For Discovery Video DNA Forensics, go to Animation and Video Files. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. Although the statistical odds of a DNA-profiling match can exceed one in 10 billion, the odds of a mistake in the collecting and testing procedures can be much greater. This is an important distinction. An error as simple as mislabeling a sample can confuse the results. Unfortunately, the odds of human error will vary and are difficult to determine.

Figure 12.15B Cheddar Man and his modern-day descendant. In Cheddar, England, DNA analysis showed a relationship between a school teacher and the remains of an ancestor, who were separated by 300 generations.

12.16 RFLPs can be used to detect differences in DNA sequences
Single nucleotide polymorphism (SNP) is a variation at one base pair within a coding or noncoding sequence Restriction fragment length polymorphism (RFLP) is a variation in the size of DNA fragments due to a SNP that alters a restriction site RFLP analysis involves comparison of sizes of restriction fragments by gel electrophoresis Restriction digests of complex genomes produce thousands of fragments with small differences in sizes that do not separate into distinct bands on a gel. To perform RFLP analysis in these cases, a method called Southern blotting is used. Southern blotting involves the transfer of DNA fragments from the gel onto a filter. The separation pattern of the fragments is preserved during the transfer. A probe or mix of probes is then used to hybridize to DNA on the filter, highlighting a subset of DNA bands for analysis. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. Here is another way to explain restriction fragment analysis. Consider these two words, equilibrium and equalibrium. Imagine that a mutation produced the spelling error of the second word. If we used a “restriction enzyme” that splits these words between u and i, how will the fragments compare in size and number? equilibrium - equ ilibri um (three fragments of three, six, and two letters) equalibrium - equalibri um (two fragments of nine and two letters)

DNA sample 1 DNA sample 2 w Cut z x Cut Cut y y Longer fragments z x w
Restriction enzymes added DNA sample 1 DNA sample 2 w Cut z x Cut Cut y y Figure RFLP analysis. This figure shows how a variation in DNA sequence alters the pattern of restriction fragments observed by gel electrophoresis. A change from a C-G base pair to an A-T base pair alters the restriction site for the DNA molecule on the right. The loss of this restriction site produces a longer DNA fragment with a size equaling the sum of two fragments from the pattern on the left. Longer fragments z x w Shorter fragments y y

12.17 Genomics is the scientific study of whole genomes
Genomics is the study of an organism’s complete set of genes and their interactions Initial studies focused on prokaryotic genomes Many eukaryotic genomes have since been investigated Evolutionary relationships can be elucidated Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast Student Misconceptions and Concerns 1. The similarities in genotypes and phenotypes among members of a human family are expected and understood by most students. Yet many students have a difficult time extrapolating this knowledge and applying it to the phylogenetic relationships of other groups. The use of genomics to test phylogenetic relationships is an enormously powerful tool for modern systematics, and genomics provides significant support of the other types of evidence for evolution. Teaching Tips 1. The first targets of genomics were prokaryotic pathogenic organisms. Consider asking your students in class to suggest why this was a good choice. Students may note that the genomes of these organisms are smaller than eukaryotes and that many of these organisms are of great medical significance.

Table Some Important Completed Genomes. This table shows the surprising estimate of 21,000 genes for humans, only 2,000 more than predicted for the nematode Caenorhabditis elegans and 4,000 fewer than the estimate for a mustard plant. Humans may be able to make a greater number of proteins with a similar number of genes, through alternative splicing mechanisms, for example. While the precise number of different types of transcripts or proteins has not been determined for humans, estimates range from 47,000 to 100,000.

The Human Genome Project revealed that most of the human genome does not consist of genes
Results of the Human Genome Project Humans have 21,000 genes in 3.2 billion nucleotide pairs Only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs The remaining 88.5% of the DNA contains Control regions such as promoters and enhancers Unique noncoding DNA Repetitive DNA Found in centromeres and telomeres Found dispersed throughout the genome, related to transposable elements that can move or be copied from one location to another Student Misconceptions and Concerns 1. The similarities in genotypes and phenotypes among members of a human family are expected and understood by most students. Yet many students have a difficult time extrapolating this knowledge and applying it to the phylogenetic relationships of other groups. The use of genomics to test phylogenetic relationships is an enormously powerful tool for modern systematics, and genomics provides significant support of the other types of evidence for evolution. 2. Students might assume that the term junk DNA implies that these noncoding regions of DNA are useless. This might be a good time to note the old saying, absence of evidence is not evidence of absence. Our current inability to understand the role(s) of noncoding DNA does not necessarily mean that these regions have no significance. 3. Students might know that humans have 23 pairs of chromosomes. Consider asking them how many different types of chromosomes are found in humans. Some will not have realized that there are 24 types, 22 autosomes plus X and Y sex chromosomes. Teaching Tips 1. The main U.S. Department of Energy Office website in support of the human genome project is found at 2. The website for the National Center for Biotechnology Information, noted in Module 12.18, is The center, established in 1988, serves as a national resource for biomedical information related to genomic data. 3. The authors note that there are 2.9 billion nucleotide pairs in the human genome. There are about 2.9 billion seconds in 91.9 years. This simple reference can add meaning to the significance of these large numbers. 4. Challenge students to explain why a complete understanding of an organism’s genome and proteomes is still not enough to understand the full biology of an organism. Ask them to consider the role of the environment in development and physiology. (One striking example of the influence of the environment is that the sex of some reptiles is determined not by the inheritance of certain chromosomes, but by incubation temperature.) 5. Students may enter your course with little appreciation of the scientific questions that remain unanswered. Struggling with the details of what we now know can overwhelm our students, leaving little room to wonder about what is not yet understood. The surprises and questions noted in Modules 12.18–12.21 reveal broad challenges that await the work of our next generation of scientists. Emphasize the many opportunities that exist to resolve unanswered questions, here and throughout your course, as an invitation to future work for students.

Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Figure Composition of the human genome. Repetitive DNA unrelated to transposable elements (15%)

Applied by J. Craig Venter and Celera vs traditional method
The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly Applied by J. Craig Venter and Celera vs traditional method Three stages of the Human Genome Project A low-resolution linkage map was developed using RFLP analysis of 5,000 genetic markers A physical map was constructed from nucleotide distances between the linkage-map markers DNA sequences for the mapped fragments were determined The first draft of the human genome sequence combined information from the three-stage map-based approach and the whole-genome shotgun method. Student Misconceptions and Concerns 1. The similarities in genotypes and phenotypes among members of a human family are expected and understood by most students. Yet many students have a difficult time extrapolating this knowledge and applying it to the phylogenetic relationships of other groups. The use of genomics to test phylogenetic relationships is an enormously powerful tool for modern systematics, and genomics provides significant support of the other types of evidence for evolution. Teaching Tips 1. Challenge students to explain why a complete understanding of an organism’s genome and proteomes is still not enough to understand the full biology of an organism. Ask them to consider the role of the environment in development and physiology. (One striking example of the influence of the environment is that the sex of some reptiles is determined not by the inheritance of certain chromosomes, but by incubation temperature.) 2. Students may enter your course with little appreciation of the scientific questions that remain unanswered. Struggling with the details of what we now know can overwhelm our students, leaving little room to wonder about what is not yet understood. The surprises and questions noted in Modules 12.18–12.21 reveal broad challenges that await the work of our next generation of scientists. Emphasize the many opportunities that exist to resolve unanswered questions, here and throughout your course, as an invitation to future work for students.

Venter’s method Whole-genome shotgun method
Whole-genome shotgun method Restriction enzymes were used to produce fragments that were cloned and sequenced Computer analysis assembled the sequence by aligning overlapping regions The first draft of the human genome sequence combined information from the three stage map-based approach and the whole-genome shotgun method. Student Misconceptions and Concerns 1. The similarities in genotypes and phenotypes among members of a human family are expected and understood by most students. Yet many students have a difficult time extrapolating this knowledge and applying it to the phylogenetic relationships of other groups. The use of genomics to test phylogenetic relationships is an enormously powerful tool for modern systematics, and genomics provides significant support of the other types of evidence for evolution. Teaching Tips 1. Challenge students to explain why a complete understanding of an organism’s genome and proteomes is still not enough to understand the full biology of an organism. Ask them to consider the role of the environment in development and physiology. (One striking example of the influence of the environment is that the sex of some reptiles is determined not by the inheritance of certain chromosomes, but by incubation temperature.) 2. Students may enter your course with little appreciation of the scientific questions that remain unanswered. Struggling with the details of what we now know can overwhelm our students, leaving little room to wonder about what is not yet understood. The surprises and questions noted in Modules 12.18–12.21 reveal broad challenges that await the work of our next generation of scientists. Emphasize the many opportunities that exist to resolve unanswered questions, here and throughout your course, as an invitation to future work for students.

Chromosome Chop up with restriction enzyme DNA fragments Sequence
Chromosome Chop up with restriction enzyme DNA fragments Sequence fragments Figure The whole-genome shotgun method. Teaching Tips Align fragments Reassemble full sequence

12.20 Proteomics is the scientific study of the full set of proteins encoded by a genome Proteomics Studies the proteome, the complete set of proteins specified by a genome Investigates protein functions and interactions The human proteome may contain 100,000 proteins. Given alternative splicing and post-translational modifications, the diversity is quite large. Student Misconceptions and Concerns 1. The similarities in genotypes and phenotypes among members of a human family are expected and understood by most students. Yet many students have a difficult time extrapolating this knowledge and applying it to the phylogenetic relationships of other groups. The use of genomics to test phylogenetic relationships is an enormously powerful tool for modern systematics, and genomics provides significant support of the other types of evidence for evolution. Teaching Tips 1. Challenge students to explain why a complete understanding of an organism’s genome and proteomes is still not enough to understand the full biology of an organism. Ask them to consider the role of the environment in development and physiology. (One striking example of the influence of the environment is that the sex of some reptiles is determined not by the inheritance of certain chromosomes, but by incubation temperature.) 2. Students may enter your course with little appreciation of the scientific questions that remain unanswered. Struggling with the details of what we now know can overwhelm our students, leaving little room to wonder about what is not yet understood. The surprises and questions noted in Modules 12.18–12.21 reveal broad challenges that await the work of our next generation of scientists. Emphasize the many opportunities that exist to resolve unanswered questions, here and throughout your course, as an invitation to future work for students.

Genomes hold clues to the evolutionary divergence of humans and chimps
Comparisons of human and chimp genomes Differ by 1.2% in single-base substitutions Differ by 2.7% in insertions and deletions of larger DNA sequences Human genome shows greater incidence of duplications Genes showing rapid evolution in humans Genes for defense against malaria and tuberculosis Gene regulating brain size FOXP2 gene involved with speech and vocalization Student Misconceptions and Concerns 1. The similarities in genotypes and phenotypes among members of a human family are expected and understood by most students. Yet many students have a difficult time extrapolating this knowledge and applying it to the phylogenetic relationships of other groups. The use of genomics to test phylogenetic relationships is an enormously powerful tool for modern systematics, and genomics provides significant support of the other types of evidence for evolution. Teaching Tips 1. Students may enter your course with little appreciation of the scientific questions that remain unanswered. Struggling with the details of what we now know can overwhelm our students, leaving little room to wonder about what is not yet understood. The surprises and questions noted in Modules 12.18–12.21 reveal broad challenges that await the work of our next generation of scientists. Emphasize the many opportunities that exist to resolve unanswered questions, here and throughout your course, as an invitation to future work for students.

How different are we?

Wrap up and Review PCR Recombinant DNA and Plasmid cloning
DNA Fingerprinting and Profiling DNA sequenceing

Bacterial clone Cut Bacterium DNA fragments Recombinant DNA plasmids Cut Recombinant bacteria Plasmids Genomic library

Mixture of DNA fragments Longer fragments move slower A “band” is a collection of DNA fragments of one particular length Power source Shorter fragments move faster DNA attracted to + pole due to PO4– groups

(a) (b) (c) (d) (e) (f) DNA amplified via Bacterial plasmids DNA
Bacterial plasmids DNA sample treated with treated with (b) DNA fragments sorted by size via (c) Recombinant plasmids are inserted into bacteria Add (d) Particular DNA sequence highlighted are copied via (e) (f) Collection is called a

(a) (b) DNA amplified via Bacterial plasmids DNA sample treated with
DNA amplified via (a) Bacterial plasmids DNA sample treated with treated with (b)

(b) (c) (d) (e) (f) DNA fragments sorted by size via
(b) DNA fragments sorted by size via (c) Recombinant plasmids are inserted into bacteria Add (d) Particular DNA sequence highlighted are copied via (e) Collection is called a (f)

You should now be able to
Distinguish between terms in the following groups: restriction enzyme—DNA ligase; GM organism—transgenic organism; SNP—RFLP; genomics—proteomics Define the following terms: cDNA, gel electrophoresis, gene cloning, genomic library, “pharm” animal, plasmid, probe, recombinant DNA, repetitive DNA, reverse transcriptase, STR, Taq polymerase, vector, whole-genome shotgun method Describe how genes are cloned

Describe how a probe is used to identify a gene of interest Describe how gene therapy has been attempted and identify challenges to the effectiveness of this treatment approach Distinguish between the use of prokaryotic and eukaryotic cells in producing recombinant DNA products Identify advantages to producing pharmaceuticals with recombinant DNA technology

Describe the basis for DNA profiling and explain how it is used to provide evidence in forensic investigations Explain how PCR provides copies of a specific DNA sequence Identify ethical concerns related to the use of recombinant DNA technology Describe how comparative information from genome projects has led to a better understanding of human biology

DNA Technology and Genomics

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DNA Technology and Genomics

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Presentation on theme: "DNA Technology and Genomics"— Presentation transcript:

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About project

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